A r
apid
and f
acile t
andem solvent solid ph
ase extr
action method w
as est
ablished to isol
atethe heterocyclic
arom
atic
amines (HAAs) 2-
amino-3,8-dimethylimid
azo[4,5-
f]quinox
aline (8-MeIQx), 2-
amino-3,4,8-trimethylimid
azo[4,5-
f]quinox
aline, 2-
amino-1-methyl-6-phenylimid
azo[4,5-
b]pyridine,
and 2-
amino-9
H-pyrido[2,3-
b]indole from urine. The HAAs were sep
ar
ated byreversed ph
ase liquid chrom
atogr
aphy
and qu
antified by electrospr
ay ioniz
ation t
andem m
assspectrometry (ESI/MS/MS) using selected re
action monitoring. The limits of detection
andqu
antit
ation of these HAAs
appro
ached 1-3
and 2-8 pg/mL, respectively, using only 0.3 mLof urine for
an
alysis. Full product ion spectr
a were
acquired to corrobor
ate
an
alyte identities.The pretre
atment of urine from hum
an volunteers th
at h
ad consumed
a grilled beef me
al with
acid or b
ase
at 70
![](/im<font color=)
ages/entities/deg.gif">C incre
ased the concentr
ation of HAAs by
as much
as 6-fold, indic
ating thepresence of ph
ase II con
jug
ates of the p
arent compounds. HAAs cont
aining
an
N-methylimid
azole moiety undergo f
acile cle
av
age of the
N-methyl group under collision-induced dissoci
ationconditions,
and MS/MS
an
alysis in the const
ant neutr
al loss sc
an mode monitoring thetr
ansition [M + H]
+ ![](/im<font color=)
ages/entities/r
arr.gif"> [M + H - CH
3
ages/entities/bull.gif">]
+ reve
aled the presence of two other HAAs. 2-Amino-3-methylimid
azo[4,5-
f]quinox
aline (IQx) w
as identified by coelution of the
an
alyte with syntheticIQx
and by
acquisition of the product ion spectrum. The second HAA w
as present in
a rel
ativelyhigh
abund
ance in urine. The molecule h
ad the s
ame nomin
al m
ass
as 8-MeIQx (MH
+ at
m/
z214),
and the product ion spectrum w
as simil
ar to th
at of 8-MeIQx. This novel HAA w
as
alsofound in the grilled me
at consumed by the volunteers
at
a concentr
ation of 8 p
arts per billion.The
accur
ate m
ass me
asurement
and product ion spectrum of this molecule by ESI qu
adrupoletime-of-flight m
ass spectrometry reve
aled th
at it w
as
an isomer of 8-MeIQx. This t
andemsolvent solid ph
ase extr
action LC/ESI/MS/MS procedure m
ay be used to r
apidly
assess thed
aily exposure to
a v
ariety of HAAs in urine.